The lack of the ability to detect odors is a very real handicap. Central to the understanding of the olfactory system is determining how the inputs, the primary olfactory sensory neurons (OSNs) function. Our long-term goal is to understand how olfactory neurons function. Studies conducted in a cyclic nucleotidegated channel (CNG) knockout mouse model, suggest that the CNG channel is involved in both the excitatory and inhibitory responses (Brunet, Gold et al. 1996; Delay and Restrepo 1999). Since the Ca-activated CI- channel is the only known downstream element in the cAMP/CNG channel pathway, this suggests that the CI current may mediate both types of responses. This would be possible if the reversal potential of chloride (Ecl) was different in individual OSNs. Thus determining the level of intracellular CI, [CI]i, and the dynamic changes in [CI]i with odor stimulation is critical to understanding the role CI plays in odor transduction. Our general hypothesis is that intracellular CI- regulation is essential to the function of olfactory neurons. Aim 1. Characterize [CI]i under resting conditions and with odor stimulation. Our hypothesis predicts that chloride levels will vary among individual OSNs and that the level of intracellular chloride will determine the response of OSNs to cAMP mediated odors. We will characterize resting [CI]i levels in isolated OSNs with optical imaging using a CI sensitive dye. Aim 2. Establish whether two chloride ion transporters, an isoform of the Na/K/2CI cotransporter, NKCC1, and an isoform of the K/CI cotransporter, KCC2, are expressed in OSNs and if there is localized expression. Determine the effects of altering the activity of NKCC1 and KCC2 on [CI]i in resting and odor-stimulated neurons. The second prediction to be tested is that specific Cl- cotransporters are expressed in OSNs and altering the function of these transporters will change the levels of [CI]i. We will focus on NKCC1 and KCC2 since these CI co-transporters have been shown to be critical to [CI]i in developing neurons and, as shown in our preliminary data, appear to be present in olfactory tissue. Aim 3. Determine the correlation between changes in [CI]i and electrophysiological responses. The third prediction to be tested is that different levels of intracellular chloride will directly affect the electrophysiological response of OSNs to odorants. We will combine optical measurements of [CI]i with patch clamp recording to determine whether the flux of CI- is correlated with the electrical responses of the OSN.